PROJECT SUMMARY/ABSTRACT Homologous recombination, i.e., homology-directed repair (HDR), is a major repair pathway for double-strand breaks (DSBs) including lesions arising during DNA replication. HDR mutants are characterized by genomic instability and sensitivity to DNA damaging agents such as inter-strand crosslinkers and poly (ADP-ribose) polymerase inhibitors. Several proteins central to the HDR pathway are tumor suppressors, notably the breast cancer suppressor BRCA2, which promotes the function of RAD51, the critical protein for homologous strand exchange. RAD51 paralogs are also tumor suppressors. The premise of this project is based on the established link of HDR with tumor suppression and therapy response. Thus, this proposal seeks to define roles of HDR factors and pathways, and the cellular responses to HDR deficiencies. The specific aims are: 1. To determine the cellular responses to RAD51 paralog deficiency. We constructed isogenic human mammary epithelial cell lines with conditional mutations in genes for the five canonical RAD51 paralogs. We plan to compare these mutants for repair of spontaneous and induced DNA damage and replication fork protection. We hypothesize that their deficiency will lead to replication stress due to HDR deficiency. 2. To determine the cellular responses to BRCA2 deficiency, including the innate immune response and factors that affect the response. BRCA2 loss leads to severe replication stress and subsequent mitotic abnormalities. We propose to determine the effect of BRCA2 deficiency on the innate immune response that senses cytosolic DNA involving cGAS-STING. Factors that may intensify or abrogate the response to replication stress will be investigated. 3. To investigate the requirement for BRCA2 DNA binding. The most conserved region of BRCA2 is the DSS1and DNA-Binding Domain. We will investigate the role of this domain in cells and mice using a number of genetic and biochemical approaches. 4. Requirement for DSS1/SEM1 for survival and DNA repair in the context of BRCA2. The interaction with DSS1 is critical for BRCA2 function in the context of the full-length protein. We will address whether DSS1 is essential solely due to its interaction with BRCA2 or whether the requirement for DSS1 is through a BRCA2- independent role.